Chapter 2Where Statistics and Molecular MicroarrayExperiments Biology MeetDiana M. KelmanskyAbstractThis review chapter pr...
16         D.M. Kelmansky                            mutual coordination with one another and with the environment.       ...
2 Where Statistics and Molecular Microarray Experiments Biology Meet                     17                          Deoxy...
18         D.M. Kelmansky                                                                5’ end                           ...
2 Where Statistics and Molecular Microarray Experiments Biology Meet                        19                            ...
20          D.M. Kelmansky                             that is replaced by Uracil (U) and the sugar is ribose instead of  ...
2 Where Statistics and Molecular Microarray Experiments Biology Meet    21            Number of expressed gene copies     ...
22        D.M. Kelmansky                           on the microarray. The microarray works as a detector of the amount    ...
2 Where Statistics and Molecular Microarray Experiments Biology Meet       233.2.1. Transcriptomic           The DNA immob...
24         D.M. Kelmansky3.4. Sample Genetic         As we have already seen, the normal cellular modification of mRNAMater...
2 Where Statistics and Molecular Microarray Experiments Biology Meet       25                                  The initial...
26         D.M. Kelmansky                                Adjacent duplicate spots (two or more times) are included to     ...
2 Where Statistics and Molecular Microarray Experiments Biology Meet        27                           A microarray imag...
28   D.M. Kelmansky                          Mk = log2 (Y1k/Y2k) in the y-axes.                          Ak = 0.5 log2 (Y1...
2 Where Statistics and Molecular Microarray Experiments Biology Meet       297. StatisticalAnalysis7.1. Inference         ...
30      D.M. Kelmansky                              Supervised classification procedures need an independent cross         ...
2 Where Statistics and Molecular Microarray Experiments Biology Meet        31                            gene expression ...
32         D.M. Kelmansky                                       Number of publications from PubMed                     600...
2 Where Statistics and Molecular Microarray Experiments Biology Meet                   33References 1. http://www-lbit.iro...
34        D.M. Kelmansky29. Huber W, Von Heydebreck A, Sultmann H,                      Inactivation of the Snf5 tumor sup...
2 Where Statistics and Molecular Microarray Experiments Biology Meet                   3552. Schena M (2003) Microarray an...
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Statistical methods for microarray data analysis

  1. 1. Chapter 2Where Statistics and Molecular MicroarrayExperiments Biology MeetDiana M. KelmanskyAbstractThis review chapter presents a statistical point of view to microarray experiments with the purpose ofunderstanding the apparent contradictions that often appear in relation to their results. We give a briefintroduction of molecular biology for nonspecialists. We describe microarray experiments from their con-struction and the biological principles the experiments rely on, to data acquisition and analysis. The role ofepidemiological approaches and sample size considerations are also discussed. Key words: Microarray experiments, Image processing, Calibration, Statistics, Epidemiology1. Introduction This chapter is written for statisticians that are faced with the challenge of getting into the increasing area of genomics and for biologists who find that is difficult to interact with statisticians. The first difficulty that statisticians and biologists encounter is the achievement of a common interdisciplinary language. This means understanding new words with new meanings and old words with different meanings and being open to having no strictly defined concepts. Gene is a good example of a concept in the process of evolving. From classical genetics its meaning rooted in the Mendelian model of monogenic diseases “the gene for”: the gene for breast cancer, the gene for hypercholesterolemia, the gene for schizophrenia. However “the gene for” is rather the gene modification that increases the odds of a person to get a certain disease and moreover it is now known that genes act inAndrei Y. Yakovlev et al. (eds.), Statistical Methods for Microarray Data Analysis: Methods and Protocols,Methods in Molecular Biology, vol. 972, DOI 10.1007/978-1-60327-337-4_2, © Springer Science+Business Media New York 2013 15
  2. 2. 16 D.M. Kelmansky mutual coordination with one another and with the environment. The term gene is used with different semantics by the major international genomic databases (1). It was originally described as a “unit of inheritance” and it has derived to a “set of features on the genome that can produce a functional unit.” The genome of any kind of organism, including humans, is the complete information needed to build and maintain a living specimen of that organism. This information is encoded in its deoxyribonucleic acid (DNA) and ranges from a few million nucleotides for a bacterium or a few billion nucleotides for a eukaryote. Every cell of our body contains the same genetic information, but what makes the unique properties of each cell type? Only a fraction of this information is active in what is called “gene expression.” Microarrays technologies provide biologists with indirect measures of the abundance of thousands of expressed DNA sequences (cDNA) or the presence of thousands of DNA sequences in an organisms’ genome. Statistical scientists might be wondering what terms like DNA, cDNA, nucleotides, genes, genome, gene expression, and eukary- ote mean and what microarray technologies are. We will begin with a brief review of molecular biology to famil- iarize a statistical reader with many genomic terms that are fre- quently encountered in relation to microarray experiments. Also we will present statistical points of view that may help biologists towards a deeper insight of their experiments random aspects.2. A BriefIntroduction toMolecular Biology Microarray experiments are usually trying to identify genes with different expression levels (differentially expressed) among several conditions. We will present the relevant biological concepts and at the end of this section a statistical reader should understand the phrase “gene expression level.”2.1. Nucleic Acids Nucleic acids can be classified in two types:(DNA–RNA) DNA, usually presenting a double stranded nucleotide chain structure. RNA, usually having a single stranded nucleotide chain structure. The monomeric units of nucleic acids are nucleotides.2.1.1. Nucleotides Each nucleotide is composed by a ● Phosphate group. ● 5 Carbon sugar (ribose in RNA, deoxyribose in DNA). ● Nitrogenous base that can be one of the following:
  3. 3. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 17 Deoxyribonucleic acid Ribonucleic acid (DNA) (RNA) nucleotide nucleotide Phosphate Base Phosphate Base 5Ј 5Ј 1Ј 1Ј 4Ј 4Ј 3Ј 2Ј 3Ј 2Ј OH OH OHFig. 1. Nucleotides’ chemical structure. Phosphate Sugar BaseFig. 2. Nucleotide schematic representation. Purines Adenine (A) in DNA and RNA Guanine (G) in DNA and RNA Pyrimidines Cytosine (C) in DNA and RNA Thymine (T) in DNA Uracil (U) in RNA The chemical structure of two nucleotides is shown in Fig. 1 where ● The 5 carbon sugar molecule is represented by a pentagon, the carbon positions are indicated by 1¢, 2¢, 3¢, 4¢, 5¢. ● The nitrogenous base is held to the carbon in the sugar 1¢ position. ● The phosphate group is joined to the 5¢ sugar position. ● The nucleotide has a free hydroxyl group in the 3¢ position (Fig. 2).2.1.2. Polynucleotide Chain Nucleotides join giving a polynucleotide chain (Fig. 3). For both DNA and RNA the union is between the 5¢ phosphate group (–PO4) of one of the nucleotides and the 3¢ hydroxyl group (–OH) of the sugar of the other nucleotide by a phosphodiester bond.
  4. 4. 18 D.M. Kelmansky 5’ end Phosphate Sugar Base Phosphate Sugar Base 3’ end Fig. 3. A simple nucleotide chain (strand) of two bases. One end of the nucleic acid polymers has a free hydroxyl (the 3¢ end), the other end has a phosphate group (the 5¢ end). This directionality, in which one end of the DNA (or RNA) strand is chemically different than the other, is very important because DNA strands are always synthesized in the 5¢ to 3¢ direction. This has determinant implications in microarray experiments. Any nucleotide chain is identified by its bases written in their sequential order. Sequences are always written from 5¢ to 3¢ ends. For example, a nucleotide chain of 6 nucleotides (and 6 bases) can be: ACGTTA.2.1.3. Oligonucleotides Oligonucleotides or oligos are short nucleotide chains of RNA or DNA. These sequences can have 20 or less bases (or pairs when they are double stranded). 50–70 nucleotide sequences are referred as long oligonucle- otides, or simply long oligos, and play an important role in microar- ray technologies.2.2. Structures The DNA structure consists of a polynucleotide double chain (or double strand) held together by weak bonds (hydrogen bonds)2.2.1. DNA Structure between the bases according to the following complementary base pairing rules C º G (with 3 hydrogen bonds). A = T (with 2 hydrogen bonds). in accordance with James Watson and Francis Crick 1953 model. The sequence of one of the strands determines the comple- mentary sequence of the other strand. Hydrogen bonds are weaker than the phosphodiester bonds in the alternating molecules of sugar and phosphate in DNA skel- eton. These binding strength differences allow the separation of the two strands under special conditions while keeping the chain structure. Denaturation and hybridization processes that we will
  5. 5. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 19 Phosphate Molecule Deoxyribose Sugar Molecule Nitrogenous Bases 5 T 3 A C G G C T A 3 5 Weak bonds Between Bases Sugar-Phosphate Backbone Fig. 4. DNA double stranded structure. Modified from http://genomics.energy.gov/gallery/ basic_genomics/detail.np/detail-14.html. see in Subheading 3 with relation to microarray experiments are deeply related to these hydrogen bonds. Figure 4 shows a four bases DNA double chain. The hydrogen (weak) bonds between the bases are shown with broken lines and the double and triple bonds are explicitly differentiated. Also single ring pyrimidines (C, T, U) and double ring purines (A, G) can be appreci- ated as well as the 5¢ to 3¢ directions of the complementary strands. Watson and Crick model also states that the two polynucleotide strands in the DNA molecule are wounded in a double helix as a twisted ladder with a sugar phosphate skeleton in the sides and nitrogen bases in the inside as rungs. Each DNA strand is half of the ladder. In 1962 Francis Crick, James Watson, and Maurice Wilkins jointly received the Medicine Nobel prize for their 1953 DNA model based on Rosalind Franklin’s work, as a molecular biologist and crystallographer. Rosalind who died of cancer in 1958 at the age of 37 could not receive the prize.2.2.2. RNA Structure As we have mentioned, the RNA is a single stranded polynucle- otide and with the same bases as DNA except for the Thymine (T)
  6. 6. 20 D.M. Kelmansky that is replaced by Uracil (U) and the sugar is ribose instead of deoxyribose as in DNA.2.3. A Eukaryotic Cell Eukaryotic is a term that identifies a cell with a membrane-bounded nucleus in contrast with prokaryotic cells that lack a distinct nucleus (e.g., bacteria). A cell’s genome is its total DNA content. Within the cell, besides the nuclear DNA of chromosomes, there are organelles in the cytoplasm called the mitochondrion with its own DNA. We will only consider nuclear DNA.2.4. Human Genome The nucleus of every human cell contains 46 chromosomes (23 pairs). Each chromosome basically consists of a long DNA double chain of approximately 2.5 × 107 nucleotides and base pairs. Unwounded, this chain can be up to 12 cm long. The human genome consists of approximately 3 × 109 base pairs. Almost all of our cells have the same genetic information. What makes a liver cell different from a skin cell? The difference results from the fact that different genes are expressed at different levels. So 1. What is a gene? 2. What does it mean that a gene is expressed? We will call gene a DNA sequence that contains the necessary information for the synthesis of a specific product. The answer to the second question is in the following section.2.5. The Central The central dogma of molecular biology states that the informa-Dogma of Molecular tion flows from DNA to RNA and then to protein. A portion ofBiology chromosomal DNA is copied (transcription process) into a single stranded messenger RNA (mRNA) that leaves the nucleus carrying the necessary information to synthesize a protein (translation pro- cess). Any sequence (or gene) that is active in this way is called “expressed.” Although a reverse process from RNA to cDNA is possible (reverse transcription) the reverse process from protein to RNA has never been obtained.2.5.1. Transcription During the transcription process only one DNA strand is copied into a RNA. The synthesis of the single stranded RNA proceeds in its 5¢ to 3¢ direction. One strand of DNA directs the synthesis of the complementary mRNA strand. This DNA strand being tran- scribed is called the template or antisense strand. The other DNA strand is called the sense or coding strand. The RNA strand newly synthesized (primary gene transcript) contains the same informa- tion as the coding strand with the same base sequence with a U instead of a T and is complementary to the template strand.Splicing In the primary gene transcript or pre-mRNA there are segments that leave the nucleus after transcription and play an active role in the protein codification process, they are called exons. There are
  7. 7. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 21 Number of expressed gene copies 30000 10000 0 G1 G20 G41 G62 G83 G106 G131 G156 G181 GeneFig. 5. Real hypothetic expression profile. also segments called introns which are part of the transcribed mRNA that does not leave the nucleus. This RNA modification in which introns are removed and exons are joined is called splicing. The messenger RNA that leaves the nucleus only has exons and in general is shorter than the original DNA template segment; it is the mature mRNA that has suffered the capping (G), polyade- nylation (AAAA…) and splicing processes. Alternative splicing is the RNA splicing variation mechanism in which the exons of the primary gene transcript, the pre-mRNA, are separated and reconnected to produce alternative ribonucle- otide arrangements. Alternative splicing allows the synthesis of a greater variety of proteins than the originally DNA segments expressed have (2). We will not describe the translation process that directs the mRNA; however, it is important to keep in mind that the amount synthesized is relatively proportional to the amount of mRNA transcribed. It is that amount of mRNA transcribed what we call gene expression level.2.5.2. Gene Expression If we could count the number of mRNA molecules for each geneProfile in a single cell we would obtain its “real expression profile.” Figure 5 shows a “real hypothetic expression profile.”3. MicroarrayTechnologies andBasic PrinciplesThey Rely On In a microarray experiment the natural process determined by the central dogma of molecular biology is interrupted to extract mature mRNA from one or more tissues or cell lines to hybridize it (we’ll soon see what this is) to its complementary cDNA previously fixed
  8. 8. 22 D.M. Kelmansky on the microarray. The microarray works as a detector of the amount and kind of mRNA present in the interrogated sample tissue. Double stranded DNA ↓ transcription or expression Simple mRNA strand -> cDNA ↓ Microarray ↓ translation Proteín3.1. What Are DNA DNA microarrays are small (2.5 cm × 6. 2.5 cm for spotted microar-Microarrays? rays and 1.28 cm × 1.28 cm for high-density chips), solid supports onto which the thousands (10,000–1,000,000) of different DNA sequences are immobilized, or attached, at two dimensional fixed matrix locations called spots or features. Each spot contains mil- lions of “identical” sequences. ● Each spot representing a different sequence has a unique phys- ical location. ● May or may not have knowledge of the sequence. The supports can be usual glass microscope slides, silicon chips, or nylon membranes. According to different array manufacturing technologies (platforms), ● DNA is printed, spotted, or actually synthesized directly onto the support. ● Features or spots can either be approximately circles or rectangles. ● Fixed sequences—DNA, cDNA, short or long DNA oligonu- cleotides—are called probes. ● Microarrays allow one-color (channel) or two-color experiments.3.2. Types of Microarrays can be classified according to the kind of the immobi-Microarrays lized genomic sequences (probes). This is important as the probe sequences in the array identify complimentary sequences in the unknown sample genomic sequences (targets).
  9. 9. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 233.2.1. Transcriptomic The DNA immobilized to the array are complementary DNAMicroarrays sequences (cDNA) derived from known transcribed mRNA sequences or possible transcribed sequences (putative genes) for a certain type of tissue, with the purpose of measuring the amount of copies of the genes that are transcribed in a moment in the experi- mental tissue. These are called gene expression microarrays.3.2.2. Comparative In Comparative Genomic Hybridization (CGH) arrays each spotGenomic Hybridization contains DNA cloned sequences with known chromosomal loca-Array tion. This allows detecting gains and losses in chromosomes. Usually probes that map to evenly spaced loci along the entire length of the genome are printed. Also large pieces of genomic DNA can serve as the probed DNA.3.2.3. Polymorphism To detect mutations, immobilized DNA is usually from polymor-Analysis Array phic variants of a single gene. The probed sequence placed on any given spot within the array will differ from that of other spots in the same microarray, sometimes by only one (Single Nucleotide Polymorphism, or SNP) or a few specific nucleotides.3.3. Basic Principles DNA microarrays rely on the complementary rule: under adequateon Which Microarray experimental conditions, complementary single stranded nucleicExperiments Rely On have strong tendency of binding in a double stranded nucleic acid molecule. For every mRNA sequence of interest (target) a complemen- tary DNA sequence (cDNA) can be obtained to immobilize a probe for that sequence onto the solid support. The position of the probe in the array identifies the sequence.3.3.1. Nucleic Acid The chemical process by which two complementary single strandedHybridization nucleic acid chains zipper up to form a double stranded moleculeand Denaturation is called hybridization. When double stranded DNA molecules are subjected to condi- tions (pH, temperature, etc.) that disrupt their hydrogen bonds, the strands are no longer held together. This means that the strands separate as individual coils, it is then said that the double helix is denatured. The denaturation conditions differ according to the relative G + C content in the DNA. The higher the G + C content of a DNA, the higher its denaturation temperature because G–C pairs are held by three H bonds whereas A–T pairs have only two. The DNA denaturation is reversible. This process is called DNA renaturation or hybridization. Hybridization reactions can occur between any (even those coming from different species) complementary single stranded nucleic acid chains: DNA/DNA, RNA/RNA, DNA/RNA. Both denaturation and hybridization processes are important in microarray experiments.
  10. 10. 24 D.M. Kelmansky3.4. Sample Genetic As we have already seen, the normal cellular modification of mRNAMaterial includes the polyadenylation process, this is the addition of up to 200 adenine nucleotides to one end of the molecule called poly(A)3.4.1. mRNA Isolation tail. In order to isolate mRNA from a given tissue, its cells are bro- ken up and the cellular contents are exposed to beads coated with strings of thymine nucleotides. Because of adenine and thymine binding affinity the poly(A) mRNA is selectively retained on the beads while the other cellular components are washed away.3.4.2. Reverse Once isolated, purified mRNA is converted to single strandedTranscription DNA using the enzyme reverse transcriptase and is then made into a stable double stranded DNA using the enzyme DNA polymerase. DNA produced in this way is called complementary DNA (cDNA) because its sequence, at least the first strand, is complementary to that of the mRNA from which it was made, and represents only exon DNA sequences.4. MicroarrayExperiments.Generaland Specific In gene expression microarrays experiments the amount of mRNARemarks (in a given tissue at a given moment for each sequence probed in the array) is measured indirectly using dye labelled molecules in order to answer, for example: ● How gene expression differs in different cell types. ● How gene expression differs in a normal and diseased (e.g., cancerous) cell. ● How gene expression changes when a cell is treated by a drug. ● How gene expression changes when the organism develops and cells are differentiating. ● How gene expression is regulated—which genes regulate which and how. There are six general steps to follow in the microarray experiments: ● Relevant questions, statistical experiment design. ● Microarray manufacturing. ● Sample preparation and target labelling. ● Hybridization of the labelled target samples sequences to the corresponding microarray probes. ● Washing to eliminate the excess solution and reduce the nonspecific binding. ● Scanning the microarray under laser light and obtaining a digi- tal image.
  11. 11. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 25 The initial data resulting from microarray experiments are one or two digital images, depending on the microarray platform used, for every microarray. Three more data analysis steps follow: ● Image analysis. ● Calibration. ● Statistical data analysis.4.1. Design Many experimental researchers, believe that statistical issues can be of secondary importance at the early stages of the experiment and that statisticians should be incorporated at the data analysis and interpretation phase of the investigation. However, also in this research area, data analysis cannot compensate for inadequate design. For microarrays experiments it is important to remember that different experimental conditions may give different expres- sion profiles for the same biological setting (3). The proposals for experimental design- and model-based anal- ysis taking into account random variability are not new (4–6). We will only describe a few specific aspects regarding the array design and the samples design.4.1.1. Array Design The choice of the DNA probe sequences to be synthesised or spot- ted on to the array depends on the technology and the type of genes the researcher wishes to interrogate or by the cDNA libraries (collections of cDNA clones) available. For high-density in situ synthesized short oligos (25 bases) microarrays it is mainly the manufactures’ decision; however, specific custom arrays can also be ordered at higher costs. Many researchers also buy or make their own spotted cDNA or long oligos (60–70 bases) microarrays. In the design of these arrays they must decide: ● Which and where the probes will be spotted. ● Which and where the controls will be spotted. Controls are special probes included in the array, some have the purpose of evaluating the quality of the experiment and others will be used to standardise the measurements. The usual control probes are: ● Negative controls: Empty spots, buffer solution spots. ● Level controls: Spots with cDNA or oligos from different spe- cies that will not interfere with the sampled genomic sequences (i.e., bacterial DNA if mammals are studied) complementary sequences will be added to the samples (spiked in) in pre specified quantities. Their intensity values serve for calibration. ● Positive controls: “Housekeeping genes,” highly expressed genes in all samples to evaluate if the hybridization has effec- tively occurred.
  12. 12. 26 D.M. Kelmansky Adjacent duplicate spots (two or more times) are included to evaluate signal variation. However this estimation will be lower than the real meaningful between array variability for a given spot among replicated arrays.4.1.2. Sample Design When multiple microarrays are hybridized with mRNA from a sin- gle biological case we have technical replicates. These replicationsTechnical Replicates only allow measuring the variability due to measurement errors and are useful in quality-control studies.Biological Replicates Biological replicates allow the evaluation of both measurement variability and biological differences between cases. This type of replication is obtained when the sample mRNA comes from differ- ent individuals from a given species or different cell lines and is required when the aim is to make inferences about populations. Although early microarray experiments used few or no biological replicates, their necessity is now undisputed (7).Sample Size Early microarray studies (8–10) used a single “two channel” microarray or two “one channel” microarrays to identify differen- tially expressed genes. Several proposals were developed to deal with the problem (11–13). The idea behind the proposals was that the measurements on many genes, i.e., variables, could compen- sate the reduced sample size. Sizes of 2 or 3 were considered large. This point of view is changing to realize that even if technical vari- ability was eliminated it is not possible to reduce random variability inherent to biological processes and that there is no alternative to increasing samples sizes in microarray studies (14).Observational Studies In many microarray experiments sample DNA or RNA come from observational studies. Bias and confounding factors should have been considered as they are in any epidemiological study; however this has not been the practise in such experiments (15, 16) which in general lack of standard epidemiological approaches (i.e., assess- ment of chance, bias, and confounding). The advantages that microarray technology can introduce in clinical and epidemiologi- cal studies, if well established epidemiologic principles are not sacrificed in the process, is beginning to be noticed (17).5. Image Analysis The resulting data from a microarray experiment are one or two digital images for each microarray in the experiment. These microarray digital images (18) provide a snapshot of the types and quantities of molecules that have reacted during hybridization and hence were present in the sample targets.
  13. 13. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 27 A microarray image is a two-dimensional numeric representa- tion in which each value gives the mean intensity of a small sector or pixel. Every spot is represented by hundreds of pixels and we have pixel wise data. It is necessary to obtain a summary intensity measure for each spot in the array. Image processing can be divided in three tasks ● Gridding: It is the assignment of spots’ coordinates. ● Segmentation: Classification of foreground (signal) and back- ground pixels. ● Target intensity extraction: Obtaining a summary measure of the spot intensity from the foreground and background pixels. Perhaps the most critical aspect of image processing is segmen- tation; this is deciding which pixels correspond to the signal for each spot. There are four commonly used segmentation methods: fixed circle, variable circle, histogram, and adaptive shape. It has not yet been established and it is not clear if there exists an optimal method (19–23). High-density oligonucleotide spots are square, and squared regions are considered for the spot foreground and background summary measures (24). Standard image processing methods subtract background from foreground intensities to obtain the final intensity value for each pixel. This background correction gives negative signals for microar- ray images. Several proposals (19–22) deal with this drawback, as missing values are artificially generated with the usual base 2 loga- rithmic (log2) data transformation used. Image analysis including background correction methods still is an active research area. Probe wise data are the final result of the image processing stage. These are the data we are considering in subsequent sections. Data from high-density microarrays require a preliminary sum- marizing step, that of probe intensities that interrogate the same genomic sequence (25, 26).6. Data Calibration Data from microarray experiments show two types of problems that are faced through data transformations: 1. MA plots present curved structures not attributable to biologi- cal reasons. 2. Probe intensity variability is mean dependent. Let Yrk represent the intensity of probe k in array r that resulted from the image processing stage. MA plots compare the intensity of two “one channel” arrays (or the two channels of a two channel microarray) in scatter plots of
  14. 14. 28 D.M. Kelmansky Mk = log2 (Y1k/Y2k) in the y-axes. Ak = 0.5 log2 (Y1k × Y2k) in the x-axes. If the samples of both arrays (or channels of the same array) come from identical biological conditions (self–self experiment) no tendency is expected in a MA plot. However curved structures not attributable to biological reasons are usually seen in these plots. A very frequently used procedure that eliminates such struc- tures consists in subtracting, to every M value in the plot, the fit of a local smoother in the original MA plot. The data on that curve are representing the probes with no different expression between the compared arrays (27). The transformed data can be written as Ck Z rk = log 2 (Yrk ) + , (1) 2 where C k is a constant depending on the spot and the local smoother. This procedure is straight forward and flexible enough to capture most of the structures appearing in MA plots but we are forcing the data to satisfy our expectations. The data transformation given in Eq. 1 solves the first of the two problems stated at the beginning of this section and partially the second one. In relation to the mean variance dependence of microarray intensity data several authors coincide in modelling intensities through a multiplicative additive model (28, 29) Yrk = ar + br X rk e ηk +ςrk + εk + δrk , (2) where Xrk is the true intensity of spot k in array r; ar , br are con- stants and ηk , ς rk , εk , δrk are error terms. Moreover the following transformation Z rk = log(Br Yrk + Cr + (Br Yrk + Cr )2 + 1), has independently been proposed by several authors (30–32) to stabilize the variance in microarray data that satisfy the multiplica- tive additive model (2) and the array dependent constants Br , Cr are estimated from the data. This transformation is based on a qua- dratic relationship between the variance and signal strength in the original scale if the data meet the model (2). This model can also explain all structures that are found in MA plots (33). Even if only the nonrandom components are considered, the nonlinear depen- dencies can be explained and removed, giving a much simpler interpretation: the true intensities are related to the observed intensities through an affine transformation Y = a + b ·X . and different choices for the parameters of the affine transformation for different arrays give the patterns observed in MA plots (34).
  15. 15. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 297. StatisticalAnalysis7.1. Inference One of the basic statistical inference concerns is to conduct tests to decide whether the difference of two sample means provides enough evidence to decide that the population means are different. In a microarray context it comes to detect genes with mean different expression levels between two or more groups (types of tissue) that provide enough evidence to decide that the genes are differentially expressed. The problem arises from the fact that thousands of tests must be conducted, one for each genomic sequence of interest. There is a widespread agreement that multiple comparison procedures should be used. The usually recommended procedure is the false discovery rate (FDR). The argument in favour of using this error rate is based on the fact that the usual correction which is obtained by using the classical Bonferroni procedure is too restrictive resulting in a reduction in power. However the strong coregulation gene structures lead to very unstable estimation of the FDR. A Bonferroni type procedure controlling the expected value of false positives results in more stable estimates than those from FDR in comparable powers (35). Multiple comparisons can be reduced and power improved through the comparison of a priori defined subsets of genes; the subsets are tested between two biological states in what is called “gene subset enrichment analysis (GSEA)” (36). This 2003 pro- posal, that uses groups of genes that share common biological function, chromosomal location, or regulation, has been used in a number of applications (37–39) and is going through subsequent improvements (40–47).7.2. Classification Unsupervised classification is one of the first statistical techniques used in the analysis of microarray data and is one of the favourites. This method attempts to divide the data into classes without prior information (unsupervised classification) or predefined classes. It has shown some successes in finding relevant and meaningful pat- terns (48–51). However, the researcher is guaranteed to obtain gene clusters, regardless of ● Sample size. ● Data quality. ● The design of the experiment. ● Any other biological validity that is affiliated with the grouping. Unsupervised classification should be avoided, if it is inevitable, some sort of reproducibility measure should be provided. Those procedures that re-sample at case level—rather than gene level— have a reasonable performance and none is considered the best.
  16. 16. 30 D.M. Kelmansky Supervised classification procedures need an independent cross validation as the resulting prediction rules are based on a relatively small number of samples of various types of tissues containing expression data of many thousands of genes. The results of classification procedures may be representing to the data too much giving low or null predictive power; this is what is called over fitting.8. Challenges Microarray technologies generated explosive expectations related to the advances that in biology and medicine would occur in the short term (51, 52) but as results did not parallel those expectations the literature reflected the disappointments (53). As the technology of microarrays ran ahead of analysis techniques, researchers from vari- ous fields carried out their own statistics to analyze the data (54). A growing number of publications per year from the year 1995 appeared since Schena (55) presented his first work on such experi- ments. Figure 6 shows the number of microarrays publications per year (selecting in Pub Med for the keywords “microarray or microarrays”). The number of articles per year has an exponential growth from 1995 to 2001. From then until 2005 the growth is linear with about a thousand more publications every year. The value of 2006 deviates slightly below this trend and in 2007 the deviation is even further.8.1. Some Successes In the decade since the beginning of technology, there have also been successes. Patients with leukaemia were automatically and accurately classified in two main subtypes of the disease using only Number of publications from PubMed 6000 4000 2000 0 1996 1998 2000 2002 2004 2006 Year Fig. 6. Number of publications selected with “microarray or microarrays” keywords.
  17. 17. 2 Where Statistics and Molecular Microarray Experiments Biology Meet 31 gene expression levels in 1999 (56). While these forms of leukaemia were already known and well characterized, the experiment showed that the strategy could in principle reveal unknown subtypes. Further in 2001, researchers identified five patterns of gene expres- sion levels in breast cancer (57) and showed that corresponded to different types of diseases with different prognosis. More recently in 2006 a gene with a fold-change of 50 times the level of expression in cancer patients who did not respond to chemotherapy treatment in comparison to those who did respond to treatment was found (58). This gene encodes for a protein that prevents tumour cell death; blocking this protein might allow a chemotherapy response. In 2005, the US Food and Drug Administration (FDA) approved the first microarray-based clinical test. The test identifies genetic variations in two key codifying regions CYP2D6 and CYP2C19 for the cytochrome P450 enzyme that metabolizes usual drugs. This will enable doctors to personalize drug choice and dosing (59).8.2. Frustrations Also, in the decade since the advent of microarray technology a great deal of frustration has accumulated among biologists who have dedicated their efforts in following up false research direc- tions. Many articles have been discredited; scientists have difficulties in finding studies that point to something concrete and in validat- ing the results and several articles show this frustration (60–62). Several studies (63–65) have found that the list of differentially expressed genes has had very low overlap between different plat- forms. However the overlap among independent studies for the same biological question can have important improvements if coherent statistical analysis are carried out (66). Numerous discussions in the literature show a tendency to explain the glaring lack of power and instability of the results of data analysis, by a high technical level of noise in the data. The Quality Control (MAQC) Consortium project has generated public available databases that may give an answer to the referred dis- cussions (67). These technical replicates addresses the evaluation of ● Repeatability within a single site. ● Reproducibility between sites. ● Comparability between platforms. Several papers reporting the analysis of MACQ data (68–73) are showing promising results on the reliability and reproducibility of microarray technology and reflecting that the random fluctuations of gene expression signals caused by technical noise are quite low. So, how are problems explained? Fig. 7 shows the number of publications selected with “microarray or microarrays” and “statis- tics or statistical” keywords. Its striking the low number of microar- ray publications presenting statistical analysis.
  18. 18. 32 D.M. Kelmansky Number of publications from PubMed 6000 microarrays statistics and microarrays 4000 2000 0 1996 1998 2000 2002 2004 2006 YearFig. 7. Number of publications selected with “microarray or microarrays” and “statistics or statistical” keywords.9. Conclusions Microarray experiments present two main types of difficulties. The first one is microarray reliability. The MACQ project currently addresses the evaluation of the repeatability within a single site, between sites reproducibility and comparability between platforms. Although some researchers believe that experiments addressed in the MAQC project are conducted in conditions too “ideal” and that hardly reflect the real situation of many experimental labora- tories (74) and sample sizes are not large enough (75), in general the results are satisfactory (76). Moreover, this technology is con- stantly evolving and improving, so it is expected to provide better and lower budget results. The second difficulty lies in the design of the experiment and data analysis. It is important to incorporate epidemiological prin- ciples to the experiment design whenever dealing with observa- tional studies. Also, microarray research area would benefit from identical design and statistical analysis for different experiments on a given biological problem. The second aspect of this goal would be achieved if researchers made available their raw data for reanaly- sis. Even better would be if research on one topic conducted by independent labs would follow the same protocol. This, together with the reduction of the experimental costs, may increase sample sizes and thus an improve power. A deeper understanding of nor- mal biological variability and gene coregulation mechanisms could be addressed (77, 78). It is critical to the advancement of knowl- edge in molecular biology that microarrays no longer be simply used as exploratory tools.
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